We have cloned the human thyrotropin beta subunit gene and have localized a major thyroid hormone inhibitory element in the first untranslated exon of this gene using a combination of functional and binding methods. This inhibitory element appeared to be comprised of two distinct half-sites, one located at the 5' end of the exon and another toward the 3' end of the exon. These various results suggest that thyroid hormone receptors binding to at least two sites in the first exon act in conjunction to mediate T3 inhibition of TSH-beta expression. We have also demonstrated that a pituitary-specific transcription factor, Pit-1, mediates both the cyclic AMP as well as the thyrotropin-releasing hormone (TRH) induction of hTSH- beta gene transcription. This is the first example of a factor known to be important in cell-specific expression also mediating hormonal regulation of pituitary gene transcription. Finally, transgenic tumors have been established using constructs of the 5'-flanking region of the human TSH- beta gene fused to the SV40 T-antigen. From such tumors, thyrotropic cell lines have been established which should be great value in the study of TSH-beta gene expression.